Abstract

We have identified two genes, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. We suggest that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis.

Members of the PIWI/Argonaute family of proteins fall into two main classes, one named after Arabidopsis Argonaute and the other after Drosophila PIWI (1). Members of this protein family contain PAZ and PIWI domains and mediate silencing via cleavage of mRNAs (2–4) or by inhibition of translation (5, 6). These proteins are found in plants (7), yeast (8), and throughout the animal kingdom—including at least eight in humans. Some of the PIWI-class proteins have been implicated in the regulation of germ cells (9–11). Very little is known, however, about how PIWI proteins regulate germ cells and whether these proteins typically promote stem cell maintenance or differentiation. Furthermore, the types of stem cells and developmental events regulated by PIWI-class proteins remain unclear.

Here we report on the planarian S. mediterranea genes smedwi-1 and smedwi-2. We studied smedwi-1 and -2 because they encode proteins similar to the PIWI class of PIWI/Argonaute proteins (fig. S1) and are thus candidates to regulate the adult somatic stem cells (neoblasts) of planarians (12, 13). Furthermore, smedwi-1 expression resembles the distribution of planarian neoblasts (14). Planarians present a promising venue for the study of PIWI-like genes because stem cells play prominent roles in homeostasis and regeneration and because the genetic study of planarian stem cells is now possible (12, 13, 15). smedwi-1 and smedwi-2 are expressed in small cells distributed like neoblasts—that is, posterior to photoreceptors and excluded from the pharynx (Fig. 1, A to C). smedwi-expressing cells, like neoblasts, reside in the parenchyma: mesenchymal tissue excluded from the nervous system, pharynx, and gastrovascular system (Fig. 1, D and E). Neoblasts are quickly and specifically eliminated after irradiation (12). Expression of smedwi-1 and -2 was markedly reduced by irradiation, consistent with their possible expression in neoblasts (Fig. 1, F to H) (table S1).

To investigate how SMEDWI proteins regulate neoblasts, we inhibited smedwi-1 and -2 with the use of RNA interference (RNAi). RNAi affected expression specifically (fig. S2A). RNAi of smedwi-1 did not cause robust defects, but RNAi of smedwi-2 resulted in 100% penetrant defects resembling those in irradiated animals lacking neoblasts. Irradiated and smedwi-2(RNAi) animals displayed regression of tissue anterior to the photoreceptors, were incapable of regeneration, and curled around their ventral surface (Fig. 2, A to C). Head regression likely occurred because tissue in front of the photoreceptors lacks dividing neoblasts and is constantly replaced by neoblast progeny (12). Because smedwi-2(RNAi) animals resemble irradiated animals, smedwi-2 is likely needed for neoblast function.

smedwi-2(RNAi) animals display defects related to neoblast dysfunction. (A to C) Irradiation at 6000 rad. C. elegans unc-22, negative control. Anterior is to the left; scale bar, 0.2 mm. Yellow arrowheads show head regression. In (B) and (C); ventral is up; in (C), heads and tails are amputated [dotted line, blastema (unpigmented) boundary]. White arrows, blastemas; yellow arrows, healed wounds without blastema. (D) Mitotic numbers divided by animal length. Left panel: Two dsRNA feedings, ≥5 animals per time point. Times shown are days after first feeding. Right panel: One dsRNA feeding, ≥6 animals per time point. Control numbers decline with time because feedings boost mitotic numbers. *P < 0.001, t test. (E) Cell numbers were analyzed by FACS (see Fig. 1I for nomenclature). Control irradiations were done 5 days before analysis. smedwi-2(RNAi) X1 and X2 irradiation-sensitive populations were present 10 days after RNAi but were greatly reduced by 18 days.

smedwi-2(RNAi) defects progress through temporal stages, ending in lethality (fig. S2B). At ≥8 days after exposure to smedwi-2 double-stranded RNA (dsRNA), animals were incapable of regeneration. Animals wounded earlier could regenerate small blastemas that regressed (fig. S2C). Homeostasis defects after smedwi-2 silencing arose with kinetics similar to those in irradiated animals. For example, 9 days after irradiation, 15 of 30 animals displayed head regression, whereas 9 days after smedwi-2 dsRNA feeding, 12 of 29 animals did so. Because irradiation prevents new cell production within 24 hours, homeostasis is likely disrupted within a day after smedwi-2 inhibition (fig. S2B). SMEDWI-2 protein levels sufficient for neoblast function in tissue turnover likely do not perdure after RNAi. This hypothesis is supported by the observation that smedwi-2 is expressed in dividing cells, which indicates that new protein is produced with new cell production.

One simple explanation for the smedwi-2(RNAi) phenotype would be absence of neoblasts. Neoblasts are the only known mitotic cells in adult planarians (12). We observed that mitotic numbers in smedwi-2(RNAi) animals always eventually plummet (Fig. 2D). We quantified this defect in intact RNAi animals. As expected, at days 11 and 14, smedwi-2(RNAi) animals had far fewer mitotic cells than did controls (P < 0.001, t test). However, smedwi-2(RNAi) animals at day 9 were defective for regeneration but had greater mitotic numbers than did the control (P < 0.001). We designed a second time course to carefully observe this increase (Fig. 2D). Because feeding boosts mitotic numbers, a single feeding was used so that early time points could be more easily observed. A clear and significant increase in mitotic numbers above the control was observed at days 7, 10, and 12 in smedwi-2(RNAi) animals (P < 0.001). In situ hybridizations with the smedwi-1 riboprobe indicated that X1 cells sharply decline in numbers rather than lose the capacity to divide at late time points after RNAi (fig. S2D). Fluorescence-activated cell sorting (FACS) analyses indicated that both X1 and X2 cells were present and irradiation-sensitive in smedwi-2(RNAi) animals incapable of regeneration 10 and 12 days after RNAi but were greatly diminished by days 14 through 18 (Fig. 2E) (fig. S3).

These experiments revealed several important aspects of the smedwi-2 phenotype. First, and in contrast to irradiated animals, neoblasts were present and proliferating at times after RNAi when animals could not regenerate. This suggests that failed neoblast maintenance was not the primary cause of failed regeneration and tissue turnover. Second, at early time points after RNAi, a greater number of smedwi-2(RNAi) mitotic neoblasts were present than in controls, suggesting a feedback regulation from loss of tissue homeostasis. Because tissue turnover fails quickly after RNAi of smedwi-2, mitotic numbers may increase during this period as a consequence of failed homeostasis. Finally, in late phases of the smedwi-2 phenotype, neoblasts are not maintained. Long-term neoblast maintenance abnormalities could be a secondary result of failed homeostasis.

We asked whether smedwi-2(RNAi) neoblasts were capable of responding to wounds in animals incapable of regeneration. Specifically, we quantified mitotic numbers in prepharyngeal animal fragments that were or were not proximal to a wound produced 14 hours earlier (Fig. 3A). We determined that smedwi-2(RNAi) animals were capable of mounting an apparently normal proliferative wounding response (Fig. 3A, P < 0.001). This finding indicates that the primary defect that blocked blastema formation in smedwi-2(RNAi) animals was not a gross dysfunction in the ability of neoblasts to detect wounds. We therefore propose that neoblast progeny function (e.g., differentiation, migration) is the primary abnormality underlying the smedwi-2(RNAi) phenotype.

smedwi-2(RNAi) neoblasts respond to wounds but produce functionally abnormal progeny cells. (A) Two prepharyngeal fragment sets were compared: anterior surface generated 14 hours before fixation in one set and 45 min before fixation in the other. *P < 0.001, t test; ≥7 fragments counted per time point. Day indicates day of analysis after first dsRNA feeding. Amputated smedwi-2(RNAi) animals regenerated abnormally by 5 days (8 of 22 animals amputated on day 7 had very small blastemas and 14 of 22 had no blastemas; 8 of 30 animals amputated on day 8 had very small blastemas and 22 of 30 had no blastemas). All controls were normal (day 7, n = 26; day 8, n = 20). (B) Cells in red are BrdU-positive 3 days after labeling. White arrowhead, tissue regression 10 days after dsRNA exposure; PR, photoreceptors. Proliferative neoblasts do not exist anterior to photoreceptors, which indicates that neoblast progeny migrated. Anterior is up; scale bar, 0.1 mm. (C) Postpharyngeal cross sections 13 days after RNAi and 6 days after BrdU labeling. White arrows, BrdU-positive cells in epidermis (outermost cells). Dotted lines are on both sides of the basement membrane of the single-cell epidermal layer. Ventral surface is up; scale bar, 10 μm.

To assess whether neoblast progeny can migrate, we labeled them with 5-bromo-2′-deoxyuridine (BrdU) and examined head tips, a region devoid of proliferating cells and into which labeled cells migrate (18). The progeny of smedwi-2(RNAi) neoblasts did not grossly fail to migrate in front of the photoreceptors of regressing heads (Fig. 3B); this result suggests that smedwi-2(RNAi) neoblasts produced cells capable of reaching sites of tissue turnover.

Next, we asked whether neoblast progeny were capable of producing differentiated cells. Neoblast progeny normally begin to replace aged epidermal cells within a week after labeling (18). We sectioned BrdU-labeled smedwi-2(RNAi) animals after BrdU incorporation and determined that labeled cells entered the epidermis sooner than in the control and displayed a grossly abnormal morphology (Fig. 3C) (fig. S4 and table S3). Planarian epidermal cells reside in a single-cell layer and display classic columnar epithelial morphology. BrdU-labeled smedwi-2(RNAi) cells that reach the epidermis, however, fail to adopt a columnar morphology. For example, the average heights of BrdU-labeled epidermal cells were significantly different in smedwi-2(RNAi) animals and control unc-22(RNAi) animals: 3.5 ± 1.6 μm (n = 18) and 10.0 ± 1.9 μm (n = 10), respectively (P < 0.0001, t test). Regions posterior to the photoreceptors, where epidermis could be clearly visualized, were observed. In smedwi-2(RNAi) animals, heads regress and the cells that reach the epidermis in the body are morphologically abnormal; for this reason, we suggest that neoblast progeny cells migrate to sites of tissue turnover but fail to function normally. That the defect is within neoblast progeny is supported by the observations that smedwi-2(RNAi) animals resemble irradiated animals and that smedwi-2 is expressed in the neoblasts.

How might a primary defect in smedwi-2(RNAi) neoblast progeny relate to the secondary defect in neoblast maintenance? Although it is unknown how an aging differentiated cell population affects neoblast division patterns, one possibility is that neoblasts are depleted because of their failure to quench an ever-increasing demand for differentiated cell replacement.

Our results provide mechanistic insight into how specific cellular events of planarian regeneration are controlled. PIWI-like proteins regulate molecular processes involving small RNAs; SMEDWI-2 may regulate some aspect of RNA metabolism within dividing neoblasts. Our data suggest that SMEDWI-2 is not needed primarily for neoblast maintenance, but rather for the production of neoblast progeny capable of replacing aged differentiated cells during homeostasis and missing tissues during regeneration. Consistent with this suggestion, the mouse PIWI-related proteins MIWI and MILI are needed for the completion of spermatogenesis but not for primordial germ cell formation (10, 11). Because human hiwi is expressed in primitive hematopoietic cells, PIWI proteins may regulate multiple types of stem cells (19). We suggest that PIWI proteins may be universal regulators of the production of stem cell progeny competent for performing differentiated functions.